energy turnover in relation to slowing of contractile properties during fatiguing contractions of...

J Physiol 587.17 (2009) pp 4329–4338 4329

Energy turnover in relation to slowing of contractileproperties during fatiguing contractions of the humananterior tibialis muscle

David A. Jones1,2, Duncan L. Turner3,4, David B. McIntyre1 and Di J. Newham5

1School of Sport and Exercise Sciences, University of Birmingham, Birmingham, UK2Institute for Biomedical Research into Human Movement and Health, Manchester Metropolitan University, Manchester, UK3School of Health and Biosciences, University of East London, London, UK4Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK5Division of Applied Biomedical Research, King’s College London, London, UK

Slowing and loss of muscle power are major factors limiting physical performance but little isknown about the molecular mechanisms involved. The slowing might be a consequence of slowdetachment of cross bridges and, if this were the case, then a reduction in the ATP cost of anisometric contraction would be expected as the muscle fatigued. The human anterior tibialismuscle was stimulated repeatedly under ischaemic conditions at 50 Hz for 1.6 s with a 50% dutycycle and muscle metabolites measured by 31P magnetic resonance spectroscopy. Over the courseof 20 contractions the half-time of relaxation increased from 36.5 ± 0.09 ms (mean ± s.e.m.) to113 ± 17 ms and isometric force was reduced to 63 ± 3% of the initial value. ATP turnoverwas determined from the change in high energy phosphates and lactate production, the latterestimated from the change of intracellular pH. ATP turnover over the first three contractions was2.45 ± 0.09 mm s−1 and decreased to 1.8 ± 0.06 mm s−1 over the last five tetani. However, whenthis latter value was normalised for the decrease in isometric force, it became 2.56 ± 0.3 mm s−1,which is the same as the turnover of the fresh muscle. The data suggest that the rate of crossbridge detachment is unaffected by fatigue and are consistent the suggestion that it is the rateof attachment which is slowed rather than the rate of detachment. The present results focusattention on stages in the cross bridge cycle concerned with attachment and the transition fromlow to high force states that may be influenced by metabolic changes in the fatiguing muscle.

(Received 10 May 2009; accepted after revision 7 July 2009; first published online 13 July 2009)Corresponding author D. A. Jones: School of Sport and Exercise Sciences, University of Birmingham, Birmingham B152TT, UK. Email: [email protected]

Abbreviations AT, anterior tibialis; MRS, magnetic resonance spectroscopy; PC, phosphocreatine; PD,phosphodiester; PM, phosphomonoester.

Metabolically demanding exercise that results in fatigueproduces shifts in contractile characteristics of the workingmuscles that are similar to the differences between fastand slow muscle fibres and this analogy could provide aninsight into the change of kinetic properties that occursduring fatigue. One feature of fast and slow muscles in thefresh state is that they have different economies of ATPturnover during isometric contractions, economy beingdefined as the ATP turnover divided by the isometric forcegenerated. The difference in economy is reported to beabout threefold in isolated animal muscles (Goldspink,1978; Crow & Kushmerick, 1982) and three- to fourfoldfor human and rat muscle fibres (Soderlund et al. 1992;Bottinelli et al. 1994; Szentesi et al. 2001). While there

are differences in the costs of calcium transport, etc., themajor part of the differences in ATP costs between differentfibre types can be ascribed to differences in cross bridgeturnover. If, therefore, fatigue is analogous to a transitionfrom a faster to a slower fibre type, the slowing of contrac-tile properties might be associated with a decrease in ATPcosts and an increase in economy of the muscle.

Edwards et al. (1975) first suggested that slow relaxationof fatigued muscle might be a consequence of slow crossbridge detachment and presented data on an isolatedmouse soleus preparation that indicated a reduction inATP turnover as the muscle fatigued and slowed. Crow &Kushmerick (1982) found no evidence of such a reductionin the soleus muscle although the experimental protocols

C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society DOI: 10.1113/jphysiol.2009.175265

4330 D. A. Jones and others J Physiol 587.17

differed in the two studies. However the latter authors didfind that the economy of the faster EDL increased over 15 sof stimulation. Subsequently they (Crow & Kushmerick,1983) showed that the change in ATP turnover in theEDL correlated with changes in the maximum velocityof unloaded shortening, although values were measuredat only two time points, 3 and 9 s. Other studies havebeen less successful in demonstrating a decrease in theenergy costs of force generation during contractions thatwould be expected to result in substantial contractileslowing. In one of the first MRS studies of skeletal muscle,Dawson et al. (1980) found no change in the economyof frog muscle as it fatigued and, likewise, Hultman &Sjostrom (1983) found no change in a biopsy study ofhuman muscle stimulated at 20 Hz over 50 s. In anotherbiopsy study of human muscle Chasiotis et al. (1987)found a tendency for economy to decrease during inter-mittent stimulation while for continuous stimulationthere was a slight increase in economy in the last stage.However, while the difference in energy cost betweenthe two protocols was significant in the latter stages,the changes with time within each protocol were notsignificant. Westra et al. (1988) compared isometric forceand ATP costs in electrically stimulated rat quadricepsand found biphasic changes with time, first an increase ineconomy and then a decrease. More recently, Giannesiniet al. (2001) measured the ATP costs of stimulating the ratgastrocnemius muscle at a range of frequencies from 0.8 to7.6 Hz and related this to the force of the evoked twitches.With protocols that induced a loss of force, the ATPcosts declined more than force and the apparent economyincreased, but there was no correlation between the extentof fatigue and change in economy and, even with theprotocols that produced no loss of force, there was stillan improvement in economy with time. There is thereforesome confusion as to whether fatigue-induced slowing ofcontractile properties is accompanied by a reduction inATP costs of the contraction.

High intensity exercise is associated with a numberof changes in contractile properties, the most evidentbeing an increased time for relaxation from an isometriccontraction (Edwards et al. 1975; Curtin, 1986, 1988;Cady et al. 1989b) and a change in the force–velocityrelationship, including a decrease in the velocity ofunloaded shortening (Crow & Kushmerick, 1983; de Haanet al. 1989; de Ruiter et al. 1999, 2000). While mostattention has focused on changes in the maximum velocityof shortening, we have shown that the time course ofthe change in relaxation rate differed from that of theslowing of shortening velocity with the change in the latterbeing a relatively late event during fatigue and playinga minor role in the characteristic loss of power (Joneset al. 2006). What was notable and unexpected in thatstudy was the fact that the curvature of the force–velocityrelationship increased substantially with fatigue and this

not only accounted for most of the loss of power butit also correlated with the change in relaxation rate. Thechange in curvature is difficult to explain in terms of slowercross bridge detachment and the authors suggested that itwas the apparent rate of cross bridge attachment whichdecreased, possibly as a consequence of increased levels ofinorganic phosphate.

A decrease in the rate of cross bridge detachment shouldbe evident as an increase in the economy of maintainingisometric force since a slowing of the rate of detachmentwould both increase force and decrease ATP turnover.On the other hand, a decrease in the rate of attachmentwould not be expected to affect economy since therewould be concomitant decreases in both force and ATPturnover. Consequently we have undertaken a study ofATP turnover in electrically stimulated human anteriortibialis (AT) muscle and related this to changes in therate of relaxation from an isometric tetanus, using thisas an indication of contractile slowing that results in aloss of muscle power. Muscle metabolites were measuredby 31P magnetic resonance spectroscopy (MRS) and theATP turnover determined and expressed as a functionof force, giving a measure of economy for the isometriccontractions.

Methods

Subjects

Four healthy subjects (one female) aged 26–49, werestudied after giving their informed consent to theexperimental protocol, which conformed to the standardsset by the latest revision of the Declaration of Helsinki andwas approved by the University College Hospital EthicsCommittee.

Force recording and electrical stimulation

Subjects lay supine with one leg extended in the boreof the MRS magnet (see below for details) and securelystrapped to a Perspex, wood and aluminium cradle thatpositioned the belly of the AT within the uniform field ofthe magnet (see Constantin-Teodosiu et al. 1997; Fig. 1).The heel rested on a small cup fixed to a hinged woodenplate and the foot and toes were firmly strapped to theupper part of the plate that was attached to an aluminiumstrain gauge. A screw arrangement between footplate andtransducer allowed adjustment of the angle of the footplate and thus optimal muscle length. The MRS coil waspositioned over the belly of the AT against the skin andadhesive ECG electrodes, used for stimulating the muscle,were placed just proximal and distal to the coil. Thestimulating current was set 20% above that required toproduce maximal tetanic force. Stimulation parameters

C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society

J Physiol 587.17 Muscle economy and slowing with fatigue 4331

were 100 ms duration square wave pulses at 50 Hz and acurrent of ∼50 mA.

Tetanic force at the end of each 1.6 s contraction wasmeasured. The relaxation rate was taken as the half-time(t0.5), in seconds, of the exponential phase (usually from50 to 25% tetanic force). The speed of relaxation was alsoexpressed as a rate constant, calculated as 0.693/t0.5.

Fatiguing procedure

The muscle was first rendered ischaemic by means of asphygmomanometer cuff placed around the thigh andinflated to 200 mmHg. A period of 30 s elapsed betweencuff inflation and the start of stimulation to deplete intra-muscular stores of O2. The ischaemic muscle was thenstimulated with 1.6 s contractions with a 1.6 s recoveryinterval between: stimulation was continued for 3, 10, 15or 20 contractions on separate occasions. At the end ofthe stimulation period the electrode leads and those to thestrain gauge were rapidly removed and muscle metabolitesdetermined whilst the muscle remained ischaemic.

Metabolite measurements

31P MRS spectra were collected before and after theischaemic exercise using methods similar to thosepreviously described by Cady et al. (1989a). Briefly, relativeconcentrations of muscle metabolites were measuredusing a spectrometer equipped with a 1.9 T super-conducting magnet and a 21 cm clear bore. A 2.1 cmtwo-turn coil was tuned for 31P (32.5 MHz) and 1H(80.3 MHz) at the centre of the magnetic field. Radiofrequency pulses giving a 90 deg flip angle at the coil centrewere used with an interpulse interval of 2.256 s. A smallsphygmomanometer cuff was placed under the calf andwas used to adjust the position of the muscle in relationto the coil, keeping it in firm contact with the skin. Theposition of the coil and whether it had moved could usuallybe judged at the end of the experiment from the positionand depth of the indentation made in the skin.

Each composite spectrum comprised 128 free inductiondecays (total collection time, 4.8 min) which weresubjected to Fourier transformation, phasing and

baseline correction. The areas for the resonance peaksof phosphomonoester (PM), inorganic phosphate (Pi),phosphocreatine (PC), phosphodiester (PD) and βATPwere calculated by integration of the area betweenpre-selected gates. The total MRS visible phosphorus wastaken to be the sum of PM + Pi + PD + PC + (3 ×βATP) and the individual compounds were expressed asa fraction of the total. Individual metabolite peaks werecorrected for partial saturation using factors derived fromcomparisons of the data obtained with the 2.256 s intervalwith that obtained with a 20.56 s interval.

Although the MRS collection time was 4.8 min there wasalways some delay in repositioning the leg and shimmingthe magnet so the time elapsed between the end ofstimulation and the end of the collection was between7 and 10 min.

βATP/total P for the resting muscles was 0.125 ± 0.007.The total P was calculated assuming the ATP concentrationto be 8.2 mM in the intracellular water (Cady et al. 1989a)giving a total P of 65.9 ± 3.7 mM. This value was then usedto estimate the absolute metabolite concentrations for thepost-exercise measurements, making the assumption thatthe total visible P signal remained constant.

Intracellular pH was estimated from the chemical shiftof the Pi peak from the PC peak:

pH = 6.75 + log[(δ − 3.27)/(5.69 − δ)]

where δ is the chemical shift (ppm) of the Pi peak relativeto PC.

Lactate production was calculated by assuming thatit was equivalent to the protons required to producethe observed change of pH, making further assumptionsabout the concentrations and dissociation constantsof the intracellular buffers. These buffers were takento be: (a) protein-bound histidine, 56 mM, pK = 6.8(Furst et al. 1970); (b) bicarbonate, 10 mM, pK = 6.1(Sahlin et al. 1977); (c) carnosine, 7 mM, pK = 6.8(Mannion et al. 1992); and (d) phosphate, added to thecomputation according to the phosphate measured byMRS (pK = 6.73). The non-phosphate buffer capacitycame to 34.5 slykes, and this increased to approximately50 slykes when Pi reached its highest levels in the fatiguedmuscles. The free intracellular ADP concentration was

Figure 1. Diagram of the experimentalarrangement for stimulating and measurement ofmetabolites from the anterior tibialis muscleSubjects lay with their leg in the bore of the magnetand the belly of the muscle positioned within theuniform magnetic field.



calculated from the concentrations of ATP, PC and H+

assuming the total creatine and phosphocreatine remainedconstant throughout the exercise at 42.5 mM:

[ADP] = ([42.5 − PC][ATP])/([PC][H+]K eq).

The equilibrium constant (K eq) for the reaction was takento be 1.66 × 10−9 M−1.

The concentration of the monobasic phosphatewas estimated from the total Pi and pH:[H2PO4

−] = ([H+][Pi])/(K p, + [H+]).The equilibrium constant K p was taken to be

1.86 × 10−7 M−1.ATP cost was estimated from changes (�;

post-contraction − pre-contraction) in PC, ATPand LA concentrations:

�ATP = (1.5 × �LA) − (�PC) − (�ATP)

Data handling

Four measurements were made for each subject so for eachthere were four determinations of resting metabolite levelsand one at the end of 3, 10, 15 and 20 tetani. The fourresting measurements for each subject were averaged toprovide a single value which was then used to determinethe change as a result of stimulation. The force recordsyielded four values for force and relaxation for each subjectfor fresh muscle and for the first three tetani, four valuesfor the next five, three for the next five, and so on. As withthe metabolites, values for each subject were averaged ateach time point to give a single value to be combined withthe data from the other subjects. ATP turnover is reportedas mM s−1, this being millimoles ATP per litre intracellularwater per second. Values are given as means and standarderrors of the mean unless stated otherwise.

Results

The stimulation protocol was well tolerated by thesubjects, the only problem being the ischaemic interval atthe end of stimulation, which became a little tedious whileadjustments were being made and MRS data collected.However the only ill effects were attacks of pins and needlesonce the blood returned to the limb.

Changes in force and contractile propertieswith fatigue

Peak force (expressed as a percentage of the value of thefirst tetanus) for the series of 20 contractions is shownin Fig. 2A. There was a suggestion of a biphasic responsewith a fairly steady decline over the first 20 s of stimulation(12–13 tetani) with a somewhat faster decline thereafter.At the end of the protocol tetanic force was reduced to63 ± 3% of the initial value.

Relaxation half-time (Fig. 2B) changed relatively littleover the first 15 s of stimulation (9–10 tetani) but thenincreased rapidly during the second half of the protocol.Values for the fresh muscle were 36.5 ± 0.9 ms and theseincreased to 113 ± 17 ms by the end of the protocol, justover a threefold increase. Expressed as rate constants,the values declined from 19 ± 0.5 s−1 in the fresh muscleto 6.7 ± 1.2 s−1 at the end the fatiguing series. Thepoint of inflection for the relaxation–time relationshipcorresponded roughly with the time at which force beganto decline more rapidly (compare Fig. 2A and B).

Muscle metabolites

Muscle metabolites measured at rest and after 3, 10, 15and 20 fatiguing tetani are given in Table 1 and shownin Fig. 3. Values are reported as concentrations in theintracellular water and are normalised to 8.2 mM in the

Figure 2. Change in contractile properties with fatigueThe ischaemic muscle was stimulated repeatedly at 50 Hz for 1.6 s with 1.6 s rest. Force (A) is expressed as apercentage of the initial isometric contraction (Po) and the rate of relaxation (B) is expressed as the half-time offorce decay.



Table 1. Muscle metabolites

Time (s) 0 4.8 16 24 32

ATP 8.2 ± 0.0 7.6 ± 0.8 7.6 ± 0.2 6.9 ± 0.7 7.0 ± 0.4PC 32.3 ± 2.4 22.2 ± 2.4 14.4 ± 1.4 9.5 ± 1.6 7.8 ± 1.6Pi 5.6 ± 0.2 14.0 ± 5.3 22.4 ± 4.3 25.4 ± 3.6 29.6 ± 2.2ME 3.3 ± 1.1 3.6 ± 0.6 6.9 ± 1.5 9.4 ± 2.2 8.4 ± 1.2pH 7.01 ± 0.02 7.09 ± 0.02 6.90 ± 0.02 6.78 ± 0.05 6.65 ± 0.05Lactate 0 0.7 ± 0.9 12.1 ± 1.1 19.4 ± 3.6 28.0 ± 3.6

The anterior tibilalis was stimulated for the times indicated and metabolites measured in theischaemic muscle by 31P MRS. Values are given as mmoles per litre intracellular water, assumingan ATP concentration of 8.2 mM in the resting muscle (0 time stimulation) and a constant totalMRS visible P thereafter. ME, phospho-monoesters. Data are given as means and S.D.

fresh muscle and, thereafter, to a constant total phosphate(see Methods).

ATP levels were unaffected for the first 10 contractionsbut thereafter declined slightly, falling to 7.0 ± 0.2 mM

from the initial value of 8.2 mM. Phosphocreatine declinedrapidly from a starting value of 32.3 ± 1.2 mM to afinal value of 7.8 ± 0.8 mM (�PC –24.5) and this wasmatched by a stochiometric rise in Pi from 5.6 ± 1 mM

to 29.6 ± 1.1 mM (�Pi 24.0). There was a rise inphospho-monoesters, due to accumulation of AMP orIMP as a consequence of the loss of ATP, but this peakwould also include sugar phosphates and other glycolyticintermediates.

Intracellular changes in hydrogen ion are shown inFig. 4A, and the corresponding pH values in Table 1. In thefirst 5 s there was a small alkalosis as a consequence of PCbreakdown, after which pH declined in a linear fashion.Resting muscle pH was 7.01 ± 0.01 which declined to6.65 ± 0.03 by the last contraction.

Lactate accumulation was calculated from the changesof intracellular hydrogen ion concentration (Fig. 4A) andthe estimated buffer capacity of the muscle and it canbe seen that there was virtually no evidence of glycolysisduring the first 5 s of contraction (3 tetani: Fig. 3), andthereafter lactate increased in a linear fashion to reach28.0 ± 1.8 mM by the last contraction.

ADP concentrations were estimated from the levels ofATP, PC and H+, assuming that the total creatine remainedconstant, and the values are given in Fig. 4B. After an initialincrease the concentration appeared to stabilise at around80 μM.

Muscle metabolites and relaxation

There were no simple relationships between change in anyof the metabolites and the slowing of relaxation, largelydue to the fact that the major change in contractile speedoccurred relatively late in the series of contractions whilethe major changes in metabolites occurred early in theprocess. The changes in total ATP were small and although

levels continued to fall throughout the series there was noincreased loss towards the end of the series that mighthave correlated with the slowing of relaxation. After aninitial fall, hydrogen ion concentration rose steadily withincreasing duration of stimulation and, at the higherconcentrations, there was a linear relationship betweenrelaxation and hydrogen ion concentration (Fig. 5A), butthis certainly did not apply over the whole pH range. Therelationship between inorganic phosphate (Pi) and rate ofrelaxation (Fig. 5B) was also biphasic with small changesin relaxation over the period when Pi was changingrapidly and more rapid slowing of relaxation in thelatter stages. Inorganic phosphate exists as the dibasic(HPO4

2−) and the monobasic form (H2PO4−), with the

proportion of the monobasic form increasing as theintracellular H+ concentration increases. The relaxationrate showed a more linear relationship with the mono-basic phosphate (Fig. 5C) than with the total Pi, althoughthe uncertainty becomes greater since the estimates of

Figure 3. Changes in ATP, phosphocreatine (PC), inorganicphosphate (Pi) and lactate (LA) estimated by 31P MRS, duringthe course of the fatiguing series of contractions illustrated inFig. 2.Concentrations are given as mmoles per litre intracellular water,assuming an ATP concentration of 8.2 mM in the resting muscle.



Figure 4. Changes in intracellular pH (A) and ADP (B) with fatigueExperimental details as described for Fig. 3.

monobasic phosphate combine the errors involved inmeasuring both Pi and H+. ADP might affect cross bridgefunction in the fatiguing muscle but it appears unlikelythat there is any causal relationship between ADP and rateof relaxation (Fig. 5) since ADP levels were constant at thetime when relaxation rates were changing rapidly.

ATP turnover

ATP turnover was calculated for each subject on the basisof change in ATP, PC and 1.5 × lactate. The changein total ATP turnover at the end of each period ofstimulation (0, 4.8, 8, 16, 24 s) was subtracted from the

Figure 5. Muscle metabolite concentrations and relaxationRelaxation rate in relation to: A, hydrogen ion concentration; B, inorganic phosphate (Pi); C, ADP; and D, monobasicphosphate (MBPi) after different periods of stimulation. Experimental details as described for Fig. 3. Data given asmeans ± S.D.



values calculated for the next longest period (4.8, 8, 16,24, 32 s respectively) and the results plotted as a meanvalue at the average time between these two times (2.4,10.4, 20 and 28 s) in Fig. 6. The initial ATP turnover was2.45 ± 0.09 mM s−1 during the first three contractions andthis declined to 1.8 ± 0.6 mM s−1 over the last 5 tetani.However it should be noted that during this time thetetanic force also declined (Fig. 2) and, if the values forATP turnover are normalised for loss of force (Fig. 7), nodecrease in turnover was evident over the course of thefatiguing contractions, the corrected turnover for the lastfive contractions being adjusted to 2.56 ± 0.8 mM s−1.

Discussion

The main purpose of this study was to determine whetherthere is a change in the economy of an isometriccontraction as the muscle fatigues and its contractileproperties become slower. A slow relaxation has beenassociated with a loss of power and this might suggest aslower rate of cross bridge detachment that would result inan improved economy. However, the observation is thatthe economy was unchanged, a result that is consistentwith the suggestion (Jones et al. 2006) that it is the rate ofcross bridge attachment that changes with fatigue.

Unlike in many animals, the human AT is a relativelyslow muscle, probably because it plays an opposing roleto the soleus muscle in maintaining an upright posture.Resting metabolite levels and contractile properties,however, were very similar to those of the hand muscles,the first dorsal interosseous (FDI) (Cady et al. 1989a) andadductor pollicis (Newham et al. 1995). The stimulatedrate of ATP turnover found in the present work ofapproximately 2.5 mM s−1 was similar to that reported

Figure 6. ATP turnoverTurnover was calculated from the data shown in Fig. 3 as the changein high energy phosphates plus the ATP from glycolysis estimated fromlactate accumulation. Values are plotted against the average time overwhich the estimates were made.

by Sacco et al. (1994), but about half the rate foundfor the adductor pollicis during a 5 s continuous tetanus(Newham et al. 1995).

The time course and extent of force loss were similar tothose seen with other human muscles fatigued in a similarfashion. The relaxation phase at the end of each tetanusslowed with the onset of fatigue, the half-relaxation timebeing three times the value in the fresh muscle. The extentof the change was somewhat less than the fourfold changereported by de Ruiter et al. (2000) and Jones et al. (2006)for the adductor pollicis, but the difference is probablyexplicable in terms of the differing stimulation protocols,since the shortening contractions used in the previousstudies would have been more energetically demandingthan the isometric contractions used in the present work.

The measurement of metabolites by in vivo 31P MRSmakes a number of assumptions and has some practicallimitations. The raw MRS data give values for thephosphorous metabolites as ratios and in convertingthese to absolute concentrations we have made twoassumptions. The first is that the intracellular ATPconcentration is 8.2 mM in the fresh muscle, while thesecond is that the total MRS visible phosphate remainsconstant throughout the series of fatiguing contractions.The first assumption will affect the absolute value ofthe ATP turnover but will not affect the conclusionthat the rate of turnover does not change with durationof activity. If the total visible P was to decrease thiswould have the effect of reducing the apparent changein PC and ATP while accentuating changes in Pi. Lactateproduction is the major determinant of ATP turnoverin the latter stages of the exercise and phosphate isa significant component of the total buffer capacity.Consequently overestimating phosphate will overestimatethe lactate production. However, any such overestimate

Figure 7. Economy of force productionEconomy of isometric force production during the fatiguing series ofcontractions estimated by normalising the data shown in Fig. 6 by theforce data in Fig. 2A.



would almost exactly be balanced by an underestimationof PC utilisation. In any case, it is unlikely that there is asignificant loss of total visible P since changes in PC and Pi

were almost exactly equal and opposite. MRS is a valuabletechnique in that it allows repeated measurements to bemade on the same muscle but has the disadvantage of beingrelatively insensitive so that a number of spectra have tobe averaged. In the system we used it was necessary tocollect spectra over 4.8 min to obtain sufficient resolutionof the major peaks. The technique is also sensitive tosmall changes in the position of the muscle in relationto the volume of the homogeneous field of the magnet.Consequently it was not possible to make concurrentforce and metabolite measurements and we adopted thetechnique of stimulating the muscle and then measuringmetabolites whilst the muscle was maintained underischaemic conditions. The assumption here is that nofurther metabolic change occurs during the time fromthe end of stimulation to collection of the final spectrum,which could be up to 10 min. However resting metabolicrates of skeletal muscle are very low compared to therates during contraction. We have found that the restingATP turnover for the adductor pollicis is of the orderof 0.01 mM s−1 compared to 5.5 mM s−1 during a tetanus(Turner, Jones & Newham, unpublished observations). Itmight be argued that the resting rate would be significantlygreater following a period of activity, but Crowther et al.(2002a,b) have shown that glycolysis turns off rapidlyat the end of contractile activity. In either case, anyresting metabolism will make an equal contribution toall our measurements and since the rates of turnoverwere calculated by subtracting one value from another,a constant additional component will not contribute tothe difference and thus the ATP turnover reported. Onefurther assumption is that the muscle was working entirelyanaerobically. Thirty seconds was allowed between makingthe muscle ischaemic and the start of the experiment,but this may not have been sufficient time to completelydeplete O2 stores in the muscle and would mean ahigher ATP turnover than was calculated. However, it islikely that any residual oxygen would be used rapidly atthe start of the contractile activity (Harris et al. 1975)and any uncertainty on this point should not affectconclusions about the economy of the longer contrac-tions when the major changes in contractile propertiesoccurred.

Although cross bridge activity may be the major processusing ATP, it is also required for calcium pumpingand maintaining electochemical gradients. The energyrequirements of these latter processes might be expected toremain relatively constant, especially if the ATP turnoveris corrected for a decrease in force, assuming this is largelythe result of a decrease in calcium release. The proportionof the total ATP consumed by cross bridge activity is notknown for the human AT muscle but it is likely to be

30–40%, as for other muscle preparations (Homsher &Kean, 1978; Crow & Kushmerick, 1983; Szentesi et al.2001). If, then, the rate of cross bridge ATP turnover wereto be halved, a reduction of perhaps 30% of the totalturnover might be expected. The data shown in Fig. 6suggest a 20% decrease in turnover, but no evidence ofa decrease is seen if a correction is made for force loss(Fig. 7).

We have not been able to measure the force–velocityrelationships of the AT muscle since this is a technicallydemanding measurement to make on human muscle insitu, especially within the NMR magnet, but it is reasonableto infer that the curvature of the relationship would havechanged in a similar way to that seen with the adductorpollicis in pervious studies where the slowing of relaxationcorrelated well with changes in curvature (Fig. 4C in Joneset al. 2006). Using the same argument, it also seems likelythat in the present experiments V max might have changedrelatively little since change in shortening velocity is asmaller and later feature of the fatigue process (Jones et al.2006).

The slowing of contractile properties with fatigue hasbeen interpreted as an indication of slow cross bridgedetachment (de Haan et al. 1989; de Ruiter et al. 1999,2000). However, the subsequent observation that changesin relaxation and loss of power were associated withan increased curvature of the force–velocity relationship(halving of a/Fo, see Jones et al. 2006) has changed thatview. In a two-state model of cross bridge action (Huxley,1957), a/Fo is determined by (f + g1)/g2, where f is the rateconstant for attachment, g1 that for detachment within thepositive working range of the cross bridge and g2 the rateconstant for detachment once the cross bridge has passedthe neutral position. The latter constant (g2) determinesV max. In the absence of a major change in V max, and thusg2, a decrease in a/Fo implies a decrease in (f + g1). Valuesfor f are generally assumed to be three- to fourfold higherthan for g1 to allow a reasonable proportion of crossbridges to be attached during an isometric contraction,and consequently it is impossible to account for a twofoldchange in (f + g1), as required to explain the reporteddecrease in a/Fo with fatigue, by changing g1 alone. Itwas argued, therefore, that to account for the changes incurvature of the force–velocity relationship with fatigue amajor change in f is required, implying a decrease in therates of cross bridge attachment rather than the previouslyheld view that it was the rate of detachment that changed.In the cross bridge model of Huxley (1957), and in morerecent models of muscle during slow shortening or inthe isometric state (e.g. Piazzesi & Lombardi, 1995), eachcross bridge cycle is associated with the hydrolysis of oneATP, so the ATP turnover per unit force in the isometricstate (economy) gives a measure of g1, the rate constantfor detachment. Our present observation of a lack ofchange in economy over the course of a series of fatiguing



contractions (Fig. 7) indicates that g1 does not change and,by implication, any decrease in a/Fo is due to a decrease inf , the rate constant for attachment.

There is then the question of whether a decrease inthe rate constant for cross bridge attachment accountsfor the slowing of relaxation from an isometric tetanus.Slow relaxation could be due to a change in cross bridgekinetics or slow Ca2+ removal from troponin into thesarcoplasmic reticulum. Although evidence from the useof mammalian preparations indicates that slowing withfatigue is primarily due to a change in cross bridge kinetics(Westerblad & Allen, 1993; Westerblad et al. 1997) this hasnot been established for human muscle working at bodytemperature. Assuming, for the moment, that cross bridgeaction does determine the rate of relaxation, it is still notimmediately obvious how a decrease in attachment mightslow the rate at which force decays. In single fibres therelaxation phase is characterised by an initial, relativelyslow, linear phase of force decline followed by a shoulderand then a more rapid exponential decay of force. Theshoulder, and subsequent loss of force, is associated withrelative movements of different elements of the fibre,generally with the end sarcomeres being stretched (Huxley& Simmons, 1970). Curtin & Edman (1989) showed thatthese differential movements are reduced in the fatiguedstate and this was associated with an increased resistanceof the fibre to stretch. Subsequently they confirmed theincreased resistance to stretch and showed that musclestiffness was increased relative to force in the fatiguedstate and suggest that this may act to stabilise the fibre.Human muscle working in situ does not show the twophases of relaxation but it does become more resistantto stretch when fatigued (de Ruiter et al. 2000). Ina two-state cross bridge model the rate constant forattachment must include a number of steps, from theactual attachment through one or more intermediary stepsto the formation of the high force state. Consequentlyan apparent decrease in the constant f could be as aresult of an increased population of cross bridges thatare attached but in low force intermediate states. Thispopulation would add resistance to stretch but reduceforce during shortening and so could stabilise sarcomerelengths and prevent stronger sarcomeres extending weakerones during relaxation, thereby slowing the decay of force.An accumulation of cross bridges in the low force statewould also have the effect of reducing curvature and thuspower output of the fatigued muscle. Thus a decrease inthe apparent rate of cross bridge attachment could explainboth slowing of relaxation and loss of power.

An increase in the concentration of inorganic phosphateis one way in which the proportion of attached crossbridges in the low force state could increase andconsequently a clear correlation might be seen betweenaccumulation of Pi and slowing of contractile properties.While the data shown in Fig. 5B do not rule this out,

they are not convincing evidence of a causal relationship.An increase in H+ might also affect cross bridge kineticsand, again, the data in Fig. 5C are inconclusive. Thecombination of Pi and H+ leads to an increase inmonobasic phosphate and the data in Fig. 5D do suggesta stronger, possibly causal, relationship with contrac-tile properties. Nevertheless, this cannot be the wholeexplanation since relaxation becomes slowed in patientswith myophosphorylase deficiency in the absence of anyincrease in H+ or monobasic Pi (Cady et al. 1989b).It is possible that relationships between changes inmetabolite levels and contractile function are obscuredby compartmentation of metabolites either within orbetween fibres.

The slowed contractile speed and loss of muscle powerseen with fatigue are major limitations to activity insport and everyday life yet very little is known about themolecular basis of these changes in function. The presentresults add weight to the suggestion that it is a reductionin the apparent rate of cross bridge attachment leading toan accumulation of cross bridges in low force states thatunderlies these changes. Attention thus focuses on factorsthat affect the stages in the cross bridge cycle concernedwith attachment and the transition from low to high forcestates as causes of contractile slowing in fatigued muscles.

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Author contributions

The authors contributed equally to the design of the study,the experimental work, subsequent analysis and drafting ofthe manuscript. The experimental work was carried out in theDepartment of Medicine, University College London.


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